1. Technical Field
The present invention relates to a method for increasing the precision of two-dimensional protein electrophoresis. More particularly, the present invention relates to a method which includes measuring the electrical conductivity of a protein sample under test, assessing the salt content of the protein sample according to the measurement result, and using a set of equations to calculate the electrical energy required respectively for protein focusing and for electrophoresis of salts in the protein sample.
2. Description of Related Art
From the 1980s onward, bioengineering has contributed significantly to the advancement of scientific techniques. In particular, electrophoretic separation, on which many important researches depend, has been an almost indispensable experimental approach in chemistry, molecular biology, and the related industries. As the net charge on a protein molecule is determined by the pH level of the surroundings, electrophoretic separation must be conducted in a special medium, typically a semi-solid gel such as polyacrylamide gel, as the matrix for electrophoresis. Nowadays, electrophoresis has evolved into a number of different techniques, namely SDS-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), two-dimensional electrophoresis, protein transfer, and so on.
In two-dimensional electrophoresis, the separation of molecules is carried out in two electrophoresis directions. More specifically, isoelectric focusing is applied to the first direction while SDS electrophoresis, which separates molecules by molecular weight, is applied to the second direction. The principles of isoelectric focusing are briefly stated as follows. To begin with, a gel containing an amphoteric electrolyte is used as the medium for electrophoresis of a protein sample. When an electrical field is applied to the gel, the amphoteric electrolyte forms a pH gradient in the gel. Under the action of the electrical field, the protein in the sample moves to a position where the pH value is equal to its isoelectric point (pI), i.e., pH=pI. Consequently, the net charge on the protein becomes zero, and the protein is focused (i.e., remains stationary) at that particular position and thus separated from the other ingredients of the sample. On the other hand, SDS-PAGE is performed in a gel added with SDS. SDS is a surfactant capable of destroying the configuration of protein molecules and evenly coating the surfaces of protein molecules with a layer of negatively charged SDS molecules. Hence, regardless of whether the protein molecules are positively or negatively charged in the first place, the SDS coating will make all the protein molecules migrate toward the positive electrode, during which process the migration speed is in inverse proportion to molecular weight. Therefore, SDS-PAGE can be used to measure the molecular weight of protein and effectuate two-dimensional protein separation in a gel.
After a biological sample under test is dissolved in a gel, the gel may contain several kinds of salts in addition to protein molecules. Due to the fact that salts have electrolytic properties, a portion of the voltage, or electrical energy, applied to the gel for protein electrophoresis is used instead to drive the electrolytic salts to migrate. As the salt content of a sample is related to the biological feature of the sample as well as the test itself and cannot be accurately evaluated, the electrical energy consumed by the salts during the isoelectric focusing process of a two-dimensional electrophoresis is hardly assessable, and so is the electrical energy used for electrophoresis of protein molecules in the sample. If isoelectric focusing is carried out with an insufficient supply of electrical energy to the protein molecules, the protein focusing effects on the isoelectric focusing gel strip will be poor, and under-focusing is likely to occur. However, an excessive supply of electrical energy may cause the protein molecules to migrate off the gel strip, thus resulting in over-focusing. Therefore, it has been a standard laboratory procedure to remove salts from a protein sample by protein precipitation and adequate rinsing, which nevertheless may lead to loss of many important proteins. Moreover, certain protein samples simply cannot precipitate effectively during the process.
The aforesaid problems have yet to be solved by persons skilled in the art.